Importance of confirming HER2 overexpression of recurrence lesion in breast cancer patients

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1 Breast Cancer (2013) 20: DOI /s ORIGINAL ARTICLE Importance of confirming HER2 overexpression of recurrence lesion in breast cancer patients Rikiya Nakamura Naohito Yamamoto Yasuhide Onai Yoshihiro Watanabe Hidetada Kawana Masaru Miyazaki Received: 7 July 2011 / Accepted: 30 January 2012 / Published online: 25 February 2012 Ó The Japanese Breast Cancer Society 2012 Abstract Background The systemic management of metastatic breast cancer (MBC) is usually based on ER or HER2 status of the primary tumor. However, the hormonal status or the overexpression of human epidermal growth factor 2 (HER2) may change in every metastatic site because of the effects of the long-term treatment of metastatic cancer with endocrine therapy, chemotherapy, or biological agents. The purpose of this study was to investigate the frequency of change in HER2 expression in primary and distant metastatic tumors in breast cancer patients. Another objective of the study was to examine the effect of the clinical therapy on the basis of HER2 expression in a metastatic tumor. Materials and methods In our hospital between 1991 to December 2010, retrospectively, 156 patients had biopsy or surgical resection of their metastatic site. All sample were analyzed pathologically to confirm metastatic disease and, second, to evaluate HER2 status by immunohistochemistry or by FISH. Results The recurrence lesions were resected from the breast or lymph node (n = 67, local lesion), brain (n = 27), lung (n = 16), liver (n = 20), bone (n = 16), and from the stomach, intestine, ovary, and uterus (n = 10). R. Nakamura (&) N. Yamamoto Y. Onai Y. Watanabe Division of Breast Surgery, Chiba Cancer Center Hospital, Nitona-Cho, Chuo-Ku, Chiba, Japan rikiya@graduate.chiba-u.jp H. Kawana Division of Diagnostic Pathology, Chiba Cancer Center Hospital, Chiba, Japan M. Miyazaki Department of General Surgery, Chiba University Graduate School of Medicine, Chiba, Japan Loss, increase, or no change in HER2 overexpression was observed in 3, 5, and 92%, respectively. Positive changes of HER2 in metastatic sites were 3 (4%) local lesion, 3 (11%) brain, 1 (7%) lung, 0 (0%) liver, 2 (17%) bone, and 0 (0%) others. In 3 of these 8 patients, trastuzumab was administered. In 2 of 3 patients, trastuzumab achieved long stable disease. The negative conversion rate of HER2 expression in metastatic lesions was 37% in patients treated with trastuzumab and 6% in those not treated with trastuzumab, a significant difference between the two groups (P \ 0.05). Conclusions The results of this study emphasize the significance of confirming HER2 expression in a recurrence lesion. For patients with positive conversion of HER2 status, more treatment options may be available. On the other hand, the rate of loss of HER2 expression was high in patients treated with trastuzumab, suggesting that the results of biopsy may provide an opportunity to reconsider treatment strategies for these patients. Keywords Introduction Metastatic breast cancer HER2 The prognosis of HER2-positive breast cancer patients has been dramatically improved by the remarkable progress in human epidermal growth factor (HER2)-targeted therapy [1]. The systemic management of metastatic breast cancer (MBC) patients is usually based on the ER or HER2 status of the primary tumor [2]. Therefore, identification of the primary tumor and whether it is HER2-positive are extremely important, and the optimum performance of the HER2 test has become a concern [3, 4]. Recent studies on breast cancer patients have indicated that the receptors of

2 Breast Cancer (2013) 20: the metastatic lesions may differ from the heterogeneous characteristics of those of the carcinoma [5, 6]. Previous studies have shown that confirmatory biopsy of metastatic cancer is important [7, 8]. However, the biopsy of the metastatic lesion is accompanied by surgical stress. Therefore, confirmation of HER2 expression in all distant metastatic lesions is a matter of debate. The effect of treatment on distant metastatic lesions, considering the lesions to be due to HER2 overexpression, is unknown. Therefore, it is necessary that we can predict there is a high possibility of a change of HER2 expression. The purpose of this study was to investigate the frequency of change in HER2 expression in primary and distant metastatic tumors in breast cancer patients. Another objective of this study was to examine the effect of the clinical therapy based on HER2 expression in a metastatic tumor. Materials and methods In our hospital between 1991 to December 2010, retrospectively, 156 patients had biopsy or surgical resection of their metastatic site. All patients were identified at the time of suspected clinical or radiological recurrence by their primary oncologist. In patients with liver and lung tumors, in particular, biopsy was indicated in the presence of an isolated tumor, and differential diagnosis between primary tumor and a metastasis from breast cancer seemed to be difficult. Biopsy was also performed for patients who received treatment for a single liver or lung metastatic lesion for at least a year and were in an advanced clinical stage. Surgical resection of brain metastases was performed as palliative therapy for patients with limited brain metastases who were in a stable general condition. Biopsy of the regional lymph node metastases was performed as much as possible to confirm the status of the biological markers. Biopsy samples were fixed in formalin and embedded in paraffin before analysis. All samples were analyzed pathologically to confirm metastatic disease and, second, to evaluate HER2 status by immunohistochemistry or by FISH. Assessment of HER2 for the metastatic tissue was compared with that for the primary tumor. The pathologist analyzing the samples was unaware of the patients original HER2 status. Immunohistochemistry All tissue samples were fixed in buffered 10% formalin and paraffin-embedded. For immunohistochemistry, all paraffin-embedded specimens were cut at 4 5 lm, by use of conventional histological techniques, and transferred to slides. Immunohistochemical staining was performed automatically with the Ventanas Benchmark Ò XT, using the Her-2/neuTest 4B5 (Ventana Medical Systems, Roche, Tokyo, Japan). HER2 overexpression was scored as weak (incomplete membrane staining in any proportion of tumor cells), 2? (complete membrane staining that is either nonuniform or weak in intensity but with obvious circumferential distribution in at least 10% of tumor cells, or invasive tumors with intense, complete membrane staining of 30% or fewer tumor cells), or 3? (uniform, intense membrane staining of [30% of invasive tumor cells) in accordance with the guidelines of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) [9]. All IHC 2? tumors were further analyzed with fluorescence in-situ hybridization to determine the HER2 gene copy level. Fluorescence in-situ hybridization (FISH) FISH analysis of the HER-2/neu gene was performed using the Histra HER2 FISH Kit (Jokoh, Tokyo, Japan). At least 60 tumor cells in each lesion and control cells were randomly evaluated for nuclear HER-2/neu amplification. Tumors were scored as amplified when the HER-2/neu gene-to-chromosome 17 ratio was C2.2 and cells had at least a median value of four HER-2/neu gene signals. The reliability of tissue microarray-based FISH for evaluation of the HER-2/neu oncogene in breast carcinoma has recently been confirmed in a validation study. Statistical analysis For statistical analysis, negative (score 0, 1, and 2 lacking amplification cases) and positive (score 2? with HER2 amplification and score 3? cases) groups were created. For correlation of data between primary tumors and metastases, the Wilcoxon signed ranks test was applied using the Superior Performing Software System (SPSS for Windows, Microsoft Japan). P values of\0.05 were considered statistically significant. Results Metastatic tumors were examined from the breast or lymph node (n = 67, local lesion), brain (n = 27), lung (n = 16), liver (n = 20), bone (n = 16), and from the stomach, intestine, ovary, and uterus (n = 10) (Table 1). Diseasefree interval for the patients is shown in Table 1. The median duration between diagnosis of the primary tumor and identification of the metastatic lesion was 1447 days (3.88 years). In many of these cases, distinction between the metastatic lesion and primary cancer was difficult. Therefore, operation or biopsy of the lesion was performed,

3 338 Breast Cancer (2013) 20: which enabled the pathologist to confirm the lesions as metastasis lesions from breast cancer. Table 2 shows the characteristics of patients with different ER or HER2 status of the primary breast cancer (PBC). We classified the study population into 4 breast cancer subtypes (Table 2): ER(?)HER2(-), ER(?)HER2(?), ER(-)HER2(?), and ER(-)HER2(-). These subtypes were observed in 46, 10, 15, and 28% of the subject population, respectively. Patients with ER(-)HER2(?) subtype of primary breast cancer were more likely to be susceptible to brain metastasis. Table 3 shows the modulation of HER2 in primary breast cancer specimens and in metastatic breast cancer specimens. Positive changes of HER2 in metastatic site were 3 (4%) local lesion, 3 (11%) brain, 1 (11%) lung, 0 (0%) liver, 1 (6%) bone, and 0 (0%) others. Negative changes of HER2 in metastatic site were 2 (3%) local lesion, 2 (10%) liver, 1 (10%) others and 0 (0%) brain, lung, and bone. The concordance rate of HER2 between PBC and MBC was 92% total. An increase in the HER2 expression was more frequent in the metastatic lesions of the brain than in the metastatic lesions of other organs. Table 4 shows the correlation of the loss of HER2 expression with trastuzumab therapy in patients with HER2-positive primary breast cancer. Forty-two HER2-positive breast cancer patients had recurrence in a variety of organs. Eight of these patients were administered trastuzumab after recurrence. In 3 of Table 1 Patients characteristics 1 Recurrence site Number (%) DFI (days) Local (breast, Ax-LN) 67 (42.9) 1475 ( ) Brain 27 (17.3) 931 ( ) Lung 16 (10.3) 1907 ( ) Liver 20 (12.8) 1279 ( ) Bone 16 (10.3) 1757 ( ) Others (intestine/ovary) 10 (6.4) 1518 ( ) Total 156 (100) 1447 ( ) Ax-LN axillary lymph node, DFI disease-free interval these 8 patients, loss of HER2 expression in the metastatic lesion was confirmed. Trastuzumab as adjuvant therapy was not administered in the remaining 34 patients. In these patients, the HER2 expression was confirmed in the metastatic site. In 2 of these 34 patients, loss of HER2 expression in the metastatic lesion was confirmed. Loss of HER2 expression in the metastatic site was significantly high in the patients treated with trastuzumab (p = 0.013). Table 5 shows the therapeutic effect after surgery of the metastatic site for HER2-positive breast cancer patients receiving trastuzumab. For these patients the metastatic lesions became resistant to trastuzumab. However, immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) of the metastatic specimens showed HER2 expression in 5 of the 8 patients. In one of the remaining 3 patients, even though HER2 expression loss was confirmed, trastuzumab was still effective. Moreover, 4 of the 5 patients were administered lapatinib, which was effective in 3 of them. Table 6 shows the response to trastuzumab therapy for patients with an increase in the HER2 expression in the metastatic lesion. Discussion The results of this study emphasize the significance of confirming HER2 expression in a metastatic lesion. Moreover, this study indicates that recurrent breast cancer should be treated on the basis of HER2 expression in the recurrence lesion. First, in some patients, the change in HER2 expression in the metastatic lesion may be attributed to the HER2- targeted therapy. Several studies have shown an increase in the HER2 overexpression in metastatic lesions [10, 11]. Fabi reported that a greater difference in the HER2 overexpression in progressive disease was observed for hormone receptor (HR)-positive cases of primary breast cancer than for HR-negative PBC [12]. However, our study indicated there was no significant difference in the effect of tamoxifen use in these cases. In our study, an increase in Table 2 Patients characteristics 2 Recurrence operation site Total no. Primary breast cancer ER(?)HER2(-) ER(?)HER2(?) ER(-)HER2(?) ER(-)HER2(-) ER estrogen receptor; HER2 human epidermal growth factor 2 Local % 8 12% 9 13% 15 23% Brain % 3 11% 9 33% 10 37% Lung % 2 13% 1 6% 7 44% Liver % 3 15% 2 10% 6 30% Bone % 1 6% 3 19% 3 19% Others % 1 10% 0 0% 3 30% Total % 18 11% 24 15% 44 28%

4 Breast Cancer (2013) 20: Table 3 Modulation of HER2 in PBC and in MBC Recurrence operation site Total no. HER2 Negative to negative positive Negative to positive negative PBC primary breast cancer, MBC metastatic breast cancer, HER2 human epidermal growth factor 2 Local % 15 22% 3 4% 2 3% Brain % 12 18% 3 11% 0 0% Lung % 3 19% 1 6% 0 0% Liver % 3 15% 0 0% 2 10% Bone % 4 25% 1 6% 0 0% Others % 0 0% 0 0% 1 10% Total % 37 24% 8 5% 5 3% Table 4 Correlation of the loss of HER2 expression with trastuzumab therapy in patients with HER2-positive primary breast cancer Received trastuzumab with HER2 positive HER2 status in MBC positive negative Yes 5 63% 3 37% 8 No 32 94% 2 6% 34 Total 37 88% 5 12% 42 P value = Total ER estrogen receptor, PR progesterone receptor, HER2 human epidermal growth factor 2 HER2 overexpression was recognized in 8 (5%) patients. In 5 of the 8 patients, trastuzumab was not administered. Unfortunately, because positive conversion of HER2 expression status in the metastatic lesions of these 5 patients was identified in this retrospective study, trastuzumab had not been used in their treatment, and therefore the potential effects of this drug remained unknown. However, in 2 of the 3 patients, trastuzumab achieved long stable disease. Our data suggest that the presence or absence of HER2 expression in the metastatic lesion in HER2-negative PBC could be confirmed by molecular target therapy. However, the effect of trastuzumab on liver and lung metastasis in 1 patient was different. Therefore, in a patient with several metastasis lesions, selection of the ideal organs for confirming the HER2 status of the tumor is complicated. Second, an alternative therapy may be administered to a patient whose metastatic lesion shows loss of HER2 expression. Our study showed that the rate of loss of HER2 expression in the metastatic lesion is higher in the group administered trastuzumab than that in the untreated group (p = 0.05). Therefore, loss of HER2 expression in the metastatic lesion may be attributed to trastuzumab therapy. However, there were no significant differences in the periods of trastuzumab use and loss of HER2 expression in the metastatic lesion. Therefore, 3 of these patients stopped receiving trastuzumab therapy and continued to receive only chemotherapy. However, one of the 3 patients resumed trastuzumab and chemotherapy because the levels of the tumor markers (carcinoembryonic antigen, serum HER2) were elevated. Another reason for resumption of trastuzumab administration for this patient was that the results of needle biopsy might not reflect the expression status of HER2 throughout the metastatic lesion because of the large size of the lesion. Third, when HER2 expression was reconfirmed in a recurrent lesion of a patient who was resistant to the agent of molecular treated therapy, an alternative molecular target reagent was recommended. In our study, trastuzumab was substituted with lapatinib for 5 patients. The conditions of 4 of the 5 patients improved after treatment with lapatinib. Lapatinib is an oral dual tyrosine kinase inhibitor that selectively inhibits the epidermal growth factor receptor (EGFR/ErbB1) and HER2/ErbB2. Clinical data have shown that for patients with HER2-positive breast cancer, lapatinib is effective as a monotherapy or in combination with trastuzumab, and in trastuzumab-resistant patients [13]. However, withdrawal of trastuzumab or chemotherapy can be clinically challenging. Confirmation of HER2 overexpression in the metastatic lesion may enable easy selection of one therapy and withdrawal of other therapy. Finally, it is important to reexamine the HER2 expression status in the primary tumor before examining the metastatic lesion, because the accuracy of examination of HER2 overexpression remains a problem. If the HER2 expression status in the primary tumor is negative, biopsy of the metastatic lesion should be considered. HER2 immunohistochemistry or HER2 FISH assays are imperfect and are less than 100% accurate and reproducible. ASCO-CAP recommends that the HER2 test must be high quality and reproducible. Our laboratory has performed this study according to a diagnosis protocol recommended in ASCO-CAP guidelines [9]. The HER2 test performed in this study shows 95% concordance with another validated test in the positive and negative assay values. In this study, we

5 340 Breast Cancer (2013) 20: Table 5 Therapeutic effect after surgery of the metastatic site for HER2-positive breast cancer patients receiving trastuzumab Case no. Metastatic site Clinical response Biopsy site HER2 change Post-treatment Clinical response 1 Liver PD Liver Negative Trastuzumab with paclitaxel SD 2 Bone PD Duodenum Negative None PD 3 Soft tissue PD Soft tissue Negative None SD 4 Breast PD Breast No change Lapatinib with capecitabin PR 5 Bone PD Bone No change Lapatinib with capecitabin SD 6 Bone PD Brain No change Lapatinib with capecitabin PD 7 Liver CR Brain No change Trastuzumab SD 8 Lung PR Brain No change Lapatinib with capecitabin PR Duration with trastuzumab: HER2-negative change versus no change: 24 versus 43 months SD stable disease, PD disease progression, PR partial, CR complete Table 6 Response to trastuzumab therapy for patients with an increase in HER2 expression in the metastatic lesion Case no. Biopsy site Primary subtype DFI Post-treatment Another metastatic sites Clinical response 1 Lung ER(-)HER2(-) 72 M Trastuzumab with Paclitaxel Lung Long SD 2 Sc LN ER(?)HER2(-) 56 M Trastuzumab with Paclitaxel Liver/lung PD/long SD 3 Inflammatory breast ER(?)HER2(-) 15 M Trastuzumab with Paclitaxel Liver PD DFI disease free interval, HER2 human epidermal growth factor 2, M month, long SD long stable disease, PD disease progression, Sc LN supraclavicular lymph node performed the HER2 test with HER2-positive and HER2- negative specimens. In addition, HER2 analysis of discordant cases by IHC was performed with FISH. Unfortunately, all HER2 tests were performed with FISH. This study is not a randomized study. However, this study demonstrated 8% discordance in HER2 status. Several studies have shown discordance between HER2 status between the primary tumor and metastases in 7 26% of cases [10, 11, 13]. In this study, the prevalence of HER2 discordance was similar if not higher than that mentioned in previous reports. Simmons demonstrated that patients were motivated to undergo biopsy to confirm their metastases, and even when the biopsy did not affect management, they reported reassurance in having tissue confirmation of their disease [11]. The purposes of treatment for metastatic breast cancer are extension of survival and improvement of quality of life. However, because of surgical stress, confirmation of HER2 expression in a recurrent lesion is impossible for many patients. In this study, the median hospital stay (range) after biopsy was: 0 (0 7 days) days for breast and regional lymph node recurrences, 1 (0 16) for liver metastases, 8 (0 22) for lung metastases, 2 (0 30) for bone metastases, and 17 (3 72) for brain metastases. A good indication for biopsy intended to examine biomarkers may be metastatic sites with minimum invasion and early resumption of treatment. In addition, some metastatic sites are resected as palliative therapy for patients with brain metastases. After the surgery, the ER and HER2 status in the resected metastatic sites should be examined. In this study, no differences were observed in the changes of the biological markers among metastatic sites. It may be important to select appropriate candidates for biopsy in future studies with a prospective design. Our study shows that molecular target agents achieved a partial response in patients who had an increase of HER-2 expression in the metastatic lesion. Before confirming the HER2 expression in a recurrent lesion, it is important to judge the indication of biopsy by considering the rate of increase in the HER2 expression and the effect of therapy. However, because of the small number of cases included in this study it is not possible to come to any definitive conclusions regarding confirmation of HER2 expression in a recurrent lesion. Further prospective studies of larger numbers of patients are required to clarify the results in greater detail. The discordances observed in HER-2 expression between primary tumor and a recurrent lesion were 8%. An increase in HER2 overexpression was recognized in 5% of patients. Use of trastuzumab or lapatinib was effective against HER2-overexpressing metastatic tumors. In this study, patients treated with trastuzumab had a tendency to lose HER2 overexpression in metastatic lesions, suggesting that biopsy of metastatic lesions may enable suitable changes in treatment strategies. Conflict of interest None declared.

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